The proposed research is a continuation of the program already under way and aims to dissect, understand and influence the function and apparent role of the cell's second genetic system, the mitochondria, in oncogenic virus-induced malignancy. Distinct differences have been found, under the current grant period, in both the structure and synthesis of mitochondrial (mt) DNA and in mitochondrial membranes, directly correlated with the phenotypic expression of oncogenic virus transformation. Nuclear DNA synthesis did not show these changes. An ideally controlled cell system of chick embryo fibroblasts infected by wild-type viruses, temperature-sensitive mutants, and non-transforming mutants of Rous sarcoma viruses (RSV) will be employed. The following parameters will be studied by isotope techniques, hybridization and electron microscopy, early after infection and after transformation during switches between permissive and non-permissive temperatures, in the presence and absence of various inhibitors: (1) Comparisons of base sequences in mt DNA's, as determined by combined restriction enzyme mapping, heteroduplex and denaturation studies; (2) mechanism of mt DNA synthesis, replication and methylation in vivo and in vitro; (3) relation to proviral DNA synthesis and integration; (4) transcription sites of mt RNA species on restriction fragments of mt DNA; (5) products of mt protein synthesis; (6) functional links between mitochondria and other parts of the cell. In addition, the expression of enucleated and fused normal/tumor cell hybrids and the expression of viral/mt DNA hybrid molecules will be studied.